Defocused reflectance imaging for low numerical aperture, large field of view quantitative live cell imaging studies

High-throughput live-cell imaging within incubator environments often necessitates a compromise between optical resolution and instrument/computational complexity. In this work, we demonstrate that controlled defocusing, a default function in all optical microscopes, can be utilized as a primary contrast mechanism for large-scale cell analysis (7000 cells per field of view). Our results suggest that even at a low numerical aperture (NA ∼ 0.01) and using just epi-illumination, defocused images can generate a uniform negative contrast across the cell body. This negative contrast improves automated cell segmentation efficiency over a wide field of view compared to in-focus imaging. We further evaluated the accessibility of this defocus imaging approach for both 2D and 3D cultures by implementing an automated cell segmentation protocol on a commercial off-the-shelf digital Universal Serial Bus (USB) microscope. The compact form factor of the digital USB microscope facilitates minimal pixel sampling, enabling high-throughput single-cell detection and continuous tracking across a large adherent cell population over several days. Our assessments of the utility of defocus imaging for 2D and 3D tissue cultures were further supported by monitoring the negative contrast changes during the migration and dissociation of 3D tissue spheroids. Our results show that negative contrast profiles from defocus images enable the quantification of cell proliferation, division, migration, and cell-to-cluster dissociation within standard culture environments. This defocusing methodology offers a scalable approach to extensive high-content screening through simplified instrumentation controls.